Somatic mutations in components of the RNA-splicing machinery (LUC7L2, PRFP8, SF3B1, SRSF2, U2AF1, ZRSR2) are detected in several subtypes of myeloid malignancies with some of them delineating a district a clinico-morphologic phenotype. RNA-splicing defects exist also in cases without splicing factor mutations suggesting that myelodysplastic syndrome (MDS) is a "spliceopathy" in which the presence of mutations represent the most extreme pole in the mis-splicing spectrum. Although some studies have shown the effects of individual splicing factor mutations on a variety of downstream targets, no key genes have been identified. Literature reports have shown that cells from patients with myeloid malignancies are highly susceptible to alterations in splicing factors with studies presenting an epistasis among RNA-splicing factor mutations (Blood.2020006868). The dilemma is whether this epistasis has a null effect or suppresses/ activates splicing factors in the context of double hits. More recently large scale genomic studies have led to the discovery of cells carrying mutations in more than one splicing factor. We took advantage of a molecular characterized collection of an in-house MDS and AML patients (n=1,742) and publicly available data of AML (n=2,302; Beat AML Master Trial, The Cancer Genome Atlas, German-Austrian Study Group) to describe the co-existence of a chimeric splicing factor phenotype and its clonal architecture. We identified a total of 40 patients carrying at least two hits in the most common components of the RNA-splicing machinery (PRPF8, SF3B1, SRSF2, U2AF1, ZRSR2). No patient harbored two hits in the same gene. In total, we detected 149 somatic mutations. A higher frequency of mutations was found in SRSF2 (60%; 24/40) followed by SF3B1 (48%; 19/40), ZRSR2 (42%; 17/40), U2AF1 (30%; 12/40) and PRPF8 (12.5%; 5/40). Other splicing factors were found mutated more rarely as in the case of LUC7L2 which was present in only 2 patients. Mutations occurred at common hotspots in SF3B1 (K666; K700; R625), SRSF2 (P95), U2AF1 (S34; Q157) and were missense, frameshift, and splice mutations in PRPF8 and frameshift in ZRSR2. Other splicing factor mutations were less frequently observed to co-occur (LUC7L2 with SF3B1 or SRSF2). In total, double splicing factor mutations were mostly enriched in male. Seventy-six% of ZRSR2 mutant patients were male. Notable the frequency of double splicing factor mutant patients was higher in AML compared to MDS and MDS/MPN (53% vs. 25% vs. 22). Thirty-two% of double mutant patients had sAML. Most common co-occurrent genes in this cohort included TET2 (32%; 13/40), RUNX1 (22%; 9/40) and ASXL1/ STAG2 (18%; 7/40 for both). The two splicing factors that were more concomitantly mutated were SF3B1/SRSF2. In fact, concomitant SF3B1/SRSF2 were found in 22% (9/40) of the patients. As a whole, SF3B1/SRSF2 average variant allele frequency (VAF)was similar (43% vs. 47%). In this group of patients, molecular hits were not preferential with half of the patients carrying one or the other as first clone. The second most concurrent mutation with SRSF2 after SF3B1 was ZRSR2 (18%; 7/40). SRSF2 was less frequently co-mutated with U2AF1 (4 patients) or PRPF8 (3 patients). U2AF1 was found co-mutated with SRSF2 and ZRSR2 (4 patients, each) and SF3B1 (3 patients). One patient with sAML had a triple splicing factor configuration (PRPF8 VAF: 29%, SF3B1 VAF: 26%, U2AF1 VAF: 20%). In terms of conventional cytogenetics, half of the patients carried normal karyotype. Among the patients with abnormal karyotype, the most common abnormality was +8 (12%; 5/40) with 4 patients having isolated +8. One patient was hemizygous carrying -17 and PRPF8 mutation. Two patients had del(7q) and del(5q) abnormalities each. The most frequent mutation in the SF3B1/SRSF2 mutant patients was RUNX1 (44%; 4/9); in two cases, SRSF2 was the dominant/ founder clone. Three patients with SF3B1/SRSF2 also carried STAG2 while two patients had TET2 and FLT3. In summary, we describe a subset of myeloid malignancies with a complex mutational associations involving splicing factor genes. The presence of these mutations might uncover novel biological clues in the mechanisms by which splicing factors drive the phenotypes of MDS and/or AML which might not represent only a random association but define new ways by which splicing factors might silence the activity of one versus another. Disclosures No relevant conflicts of interest to declare.